The present invention relates to a method, system and an article of manufacture for clustering and thereby identifying predefined binding moieties of one type which are reactive with undetermined binding moieties of a second type. More particularly, the present invention relates to a method, system and an article of manufacture for clustering and thereby identifying predefined antigens reactive with undetermined immunoglobulins of sera derived from patient subjects in need of diagnosis of disease or monitoring of treatment.
Autoimmune diseases are caused by an attack of a patient's own immune system against otherwise healthy self components of the body. Autoimmune diseases include, for example, type 1 diabetes, Behcet's disease, multiple sclerosis, rheumatoid arthritis, idiopathic thrombocytopenic purpura and various diseases affecting every organ and almost every cell type in the body. These diseases tend to run a relapsing or chronic course, and in many cases affect young individuals in the prime of life. The various autoimmune diseases are often difficult to diagnose early in their course because the clinical picture can, at times, be obscure at onset. It is even more difficult to identify incipient disease in persons at risk. Diagnosis and early diagnosis prior to accumulation of irreversible damage, is becoming more critical because specific immune therapies are now being implemented. To this end, see for example, U.S. Pat. Nos. 5,114,844; 5,671,848; 5,578,303; 5,780,034 and EP 0417271 with respect to IDDM, and Cop-1 in MS (U.S. Pat. Nos. 3,849,550; 5,800,808; 6,048,898; and 6,054,430, which is incorporated herein by reference. The earlier immune treatments are instituted, the more effective they can be.
Traditionally, immunologic diagnosis has been based on an attempt to correlate each disease with a specific immune reactivity, such as an antibody or a T-cell response to a single antigen specific for the disease entity. This approach has been largely unsuccessful for three main reasons: First, a specific antigen or antigens have not been identified for the disease, as is the case in, for example, Behcet's disease, rheumatoid arthritis (1). Second, immunity to multiple self-antigens, and not to a single self-antigen, is manifest in various patients suffering from a single disease. For example, a dozen different antigens are associated with type 1 diabetes (2). Third, a significant number of healthy individuals may manifest antibodies or T-cell reactivities to self-antigens targeted in autoimmune diseases, such as insulin, DNA, myelin basic protein, thyroglobulin and others. Hence, there is a real danger of making a false diagnosis based on the determination of a single immune reactivity. Novel approaches, therefore, are needed to support the diagnoses of specific autoimmune diseases in a way that would justify specific therapeutic interventions.
Chronic diseases that are not thought to be autoimmune are also in need of new diagnostic methods. Many chronic conditions, such as Alzheimer's disease of the brain, various dystrophies of the muscles, psoriasis of the skin, and others, involve inflammation, and one needs convenient tools to help categorize different types of inflammation. These conditions include degenerative and metabolic diseases. Inflamation is also a key factor in transplantation reactions, in healing and in tissue regeneration. The challenge is not only to diagnose the disease, but also to distinguish individuals who would benefit from a particular treatment from those individuals who would not.
Infectious diseases, too, require better diagnostic discrimination between persons who will be susceptible to a particular treatment and persons who will not respond thereto. Certain infections can trigger autoimmune responses, and it is important to be able to diagnose persons who are destined to develop autoimmune diseases.
The immunotherapy of cancer is another situation in which it would be advantageous to classify persons with different types of immune reactivities to self-antigens; many, if not most tumor-associated antigens are self-antigens. Thus, it could be important in the design of therapeutic tumor vaccines to know what kind of autoimmune reactivity is found in the patient.
The immune system regulates inflamation and the state of the immune system reflects the state of the body in many different conditions. Thus, it is evident that assays for monitoring the state of the immune system are needed. Various immunologic therapies are now being used. There is a critical need to develop markers that will enable the physician to monitor the response of the immune system to various treatments designed to arrest chronic inflammation and autoimmune diseases, vaccinate against infectious agents, or effect the immunotherapy of cancer.
Immune diagnosis and immune monitoring require ways to ascertain the state of an individual's immune system, and to record the dynamic evolution of changes induced by the various therapeutic interventions. Tools for diagnosis and monitoring are likely to require the integration of large amounts of information for the following reasons:
First, the human immune system is enormously complex and its long-term behavior is not easily explained by any particular genes or clones of cells in isolation. For example, it is now known that many autoimmune diseases involve collectives of self-antigens and collective cross-regulation. Indeed, effective tumor immunotherapy may require controlled autoimmunity, and assays for the global state of autoimmunity are therefore essential.
Second, immune system behavior depends on the state of multiple regulatory mechanisms, and not merely on the recognition of one or another antigen.
Third, individual persons, because of their genetic make-up and their varying immune histories are likely to require individualized therapies. The type, amount and schedule of immune regulation or vaccination must be tailored to the needs of the individual.
Thus, the complexity of the immune system is such that one must develop bio-informatic methods that will allow a physician to monitor conveniently the global state of the patient's immune system in health, disease and therapeutic intervention. Such a novel approach is described herein.
In the past, attempts have been made to detect the changes that the immune system undergoes in pathological conditions, with the hope that understanding such changes would lead to a better diagnosis and treatment of patients suffering from autoimmune disorders. In the simplest approach, these efforts have concentrated on the detection of specific antibodies directed to single antigens thought to be relevant to the particular disease (3). Many factors have rendered these attempts unsuccessful. Among them are the low prevalence of the studied antibody reactivities in the patient population, associated with large individual variations that can be observed among patients suffering from the same disease (4). Furthermore, natural auto-antibodies directed against the test antigens are often detected in the sera of healthy individuals (5), complicating the use of these discrete antigen-antibody methods for diagnostic purposes.
Other studies have focused on poly-reactive antibodies each able to recognize a number of different self-antigens. These poly-reactive antibodies, however, have been found both in healthy persons and in patients undergoing autoimmune or tumor-associated processes (6, 7). Therefore, several attempts were made to analyze fluctuations in the levels of auto-reactive antibodies, and changes in the repertoire of recognized antigens. These assays were mainly based on western-blotting techniques directed to simultaneously follow antibody reactivities to several auto-antigens.
The Immunoblotting and Densitometric Subtraction Method was developed as a technique for immunoblotting analysis of the reactions of natural autoantibodies in whole sera of patients (8). By densitometric subtraction, natural autoantibodies present in healthy individuals were differentiated from disease-associated autoantibodies. This method is, however, limited to a few antigens and it does not solve the problem of variation among different experiments which is inherent to blot techniques.
Another method developed to detect antibody repertoires is the Multiple Spot Immunoassay, which assays the reactivities towards 42 different antigens coated onto nitrocellulose, in a western blot procedure (9). The antibody staining in this system is analyzed, and the amount of antibody to the antigens can be semi-quantified, using IgG standards. This method allows rapid screening of auto-antibodies but does not solve the problem of auto-antibodies found in healthy persons. Moreover, it does not solve the intrinsic variation associated with the western blot technique.
Currently, the principle technique in use for assaying antibody patterns is the Panama Blot System. This too is a western-blot system, and it is based on the blotting of undefined tissue extracts. The Panama Blot employs double staining of nitrocellulose membranes to reveal both antibody reactivities and the migration position of the blotted proteins in the membrane (10, 11). This double staining allows the standardization of the results obtained for each patient. However, since the antigens used in the method are complex mixtures extracted from different tissues, they are not at all identified. Thus, this approach, even while facing the central problem of test variation, does not provide accurate information about the specific antigens recognized; it merely reveals patterns of reactivities, whose targets are totally unknown. The blots tend to vary from test to test, according to the ill-defined tissue extraction and the varying separation of the proteins. Indeed, several different antigens are undoubtedly present in each band.
As an alternative approach, some groups have studied natural auto-antibody reactivity towards panels of selected auto-antigens by means of enzyme immunoassay analysis. However, in order to get meaningful results in these studies, it was necessary to purify various antibody isotypes (12, 13). This purification step by itself, however, invalidates any possible physiological interpretation of the results because the analyzed sample does not reflect the in vivo situation, where different antibody isotypes are mixed and regulate each other (14). Therefore results obtained by this technique are deeply modified by the observer, ruling out any possible application in the management of patient treatment.
There is thus a widely recognized need for, and it would be highly advantageous to have, objective means with which one can diagnose autoimmune diseases and other diseases characterized by an inherent or induced impaired immune system, devoid of the above limitations.